In recent years, it has been discovered that N-acetyl-D-glucosamine is a valuable pharmacological agent in the treatment of a wide variety of ailments. N-acetyl-D-glucosamine does not have any established negative side effects. Rather, since it occupies an important place in protein synthesis, it has a positive effect on tissue regeneration. N-acetyl-D-glucosamine has therapeutic potential in the treatment of a wide variety of diseases including gastritis, food allergies, inflammatory bowel disease (IBD) and diverticulitis. N-acetyl-D-glucosamine is a valuable and important component of protein synthesis in the animal body.
Currently, N-acetyl-D-glucosamine is not widely available in the marketplace and is expensive. To date, N-acetyl-D-glucosamine in commercial quantities is prepared by a United States company which utilizes a process that is based on acid hydrolysis of crude chitin. This results in deacetylation of N-acetyl-D-glucosamine units to form glucosamine. The glucosamine is isolated and re-acetylated to N-acetyl-D-glucosamine using an organic acetylating reagent such as acetic anhydride.
Chitin, which is a major component of the exoskeleton of insects and shellfish, is a .beta. 1,4 linked unbranched polymer composed almost entirely of amino sugars, specifically N-acetyl-D-glucosamine, and is a natural substrate for the manufacture of this compound. N-acetyl-D-glucosamine is a natural substance, has a somewhat pleasant sweet taste, is non-toxic and dissolves easily in water and bodily fluids.
N-acetyl-D-glucosamine, specifically2-acetamido-2-deoxy-D-glucose, belongs to a larger class of amino sugars which serve a number of important functions and are localized in many areas of the human biosystem. N-acetyl-D-glucosamine is a key amino sugar which is made in the body of all animals from glucose, also called blood sugar. Complex (polymeric) carbohydrates containing N-acetyl glucosamine, referred to as glycosaminoglycans, become associated with or attached to proteins to form a class of compounds generally referred to as proteoglycans. N-acetyl-D-glucosamine is part of the makeup of body tissues and blood vessels, and the protective coverings over the digestive, respiratory and genitourinary organs. In this capacity, it is involved in the regulation of what enters and leaves the body and the movement of substances into and between cells of the body. The biochemical processes involved in amino sugar synthesis and utilization occur in all cells and are fairly well understood.
Amino sugars make up over half the glycosamino-glycans of interstitial tissue that fills the spaces between cells and forms the cellular "glue" that binds cells together. This material is a gel-like matrix of collagen protein and glycosamino-glycans, holding cells in place and regulating those substances which pass between cells.
Amino sugars are also a major constituent of basement membranes; the tough, thin sheets which surround blood vessels and various tissues. These tissues control the nutrients and waste products that cross the cell membranes, supporting tissue structures and influence cell growth.
Certain disease processes appear to be related to abnormalities in the formation and utilization of amino sugars which are natural constituents of many tissues in the body. Since amino sugars are vital components for many tissues it is easy to rationalize that a reduction in the availability of amino sugars would lead to abnormal cellular functions. Cell membranes, intercellular fluids, cell regeneration and overall tissue metabolism could all be affected. A deficiency in this fundamental building block in the body could lead to a variety of medical disorders.
In U.S. Pat. No. 5,262,310, granted Nov. 16, 1993, Isao Karube et al., discloses and claims a process wherein chitin-containing material is heat-treated in organic solvent, or in a solvent with water, and then .beta.-1,4 glycoside decomposing enzyme is added for decomposing the chitin-containing material by enzyme reaction. Alternatively, chitin-containing material is ultrasonicated in organic solvent or in the solvent with water, and then chitin-containing material is decomposed by enzyme reaction of .beta.-1,4 glycoside decomposing enzyme. Further, chitin-containing material is exposed in the solution containing urea and/or surfactant, and then chitin-containing material is let co-exist with .beta.-1,4 glycoside decomposing enzyme under the presence or non-presence of urea and/or surfactant for decomposing chitin-containing material. In a further variation, at the time of exposing chitin-containing material in the solution containing urea and/or surfactant, the solution is heated and the .beta.-1,4 glycoside decomposing enzyme is added for decomposing chitin-containing material. Specifically, the process comprises an enzymatic method of decomposing chitin, comprising: mixing the chitin with an organic solvent, thereby creating a chitin and solvent mixture, wherein the mixing step comprises ultrasonicating the chitin and solvent mixture; heating the chitin and solvent mixture; and adding .beta.-1,4 glycosidase to the mixture.
A number of Japanese patents disclose various methods of treating chitin, or chitin derivatives, with chitinase enzymes.
Japanese Patent No. 5,084,087, Apr. 6, 1993, discloses the release of N-acetyl-D-glucosamine by treating a polysaccharide with acid chitinase induced with ethylene in azuki bean plant.
Japanese Patent No. 5,007,496, Jan. 19, 1993, discloses the preparation of the oligosugar (high polymerized heterooligo sugar of glucosamine and N-acetyl glucosamine, and monooligosugar of glucosamine) by hydrolysis of the partially deacetylated chitin by chitinase. The preparation is carried out in a dialysis membrane, by dialysis. Cation ion exchange chromatography is placed outside the membrane. The chitin is obtained from crustacea or tricomonus.
Japanese Patent No. 3,094,697, Apr. 19, 1991, discloses a method comprising completely decomposing chitin or chitosan into their structural units, N-acetyl glucosamine and glucosamine using enzymes, and then determining the monosaccharide. The deacetylation degree of chitin and chitosan can be measured readily with high accuracy. The enzyme preferably includes exo type .beta.-D-glucosoaminidase, .beta.-N-acetylhexosaminidase, chitosanase, chitinase, lysozyme, etc. The measurement of N-acetyl glucosamine and glucosamine is conducted using high speed liquid chromatography, colorimetory, etc.
Japanese Patent No. 2,031,685, Feb. 1, 1990, discloses a method of producing monosaccharides characterized by (a) treating starch and/or its hydrolyzate with endo-form polysaccharide-decomposing enzyme and (b) accumulating monosaccharide with high concentration. As polysaccharide and/or its hydrolyzate starch and/or its hydrolyzate, xylan and/or its hydrolyzate, mannan and/or its hydrolyzate, chitin and/or its hydrolyzate and galactan and its hydrolyzate can be used and as polysaccharide-decomposing enzyme, alpha-amylase, xylanase, mannase, chitinase and galactanase can be used respectively. The monosaccharide formed by the above combination is glucose, xylose, mannose, N-acetyl glucosamine or galactose.
Japanese Patent No. 1,291,793, Nov. 24, 1989, discloses endo-type chitinase production by cultivation of trichoderma AF6-T8 in liquid medium containing colloidal or fine particles, used to produce acetyl glucosamine.
Japanese Patent No. 93,004,067, Jan. 19, 1993, discloses the production of chitosan, N-acetyl glucosamine, etc., by cultivating Flavobacterium MP-1C and/or Pseudomonas MP-1D strains in the presence of crustaceae shells. The chitin in the shell can purportedly be effectively decomposed and chitosan, N-acetyl-glucosamine, etc. can be prepared. Chitin-decomposing enzymes such as chitinase, chitobiase, chitin deacetylase and chitosanase can also be recovered by fractionating means such as ultrafiltration, etc.
Japanese Patent No. 93,033,037, May 18, 1993, discloses N-acetyl-D-glucosamine preparation by hydrolyzing chitin to N-acetyl-chito-oligosaccharide, and then further hydrolyzing with an enzyme. The method is characterized by (a) hydrolyzing chitin partially with acid and (b) treating the thus obtained mixture containing N-acetylchito-oligosaccharides with the enzyme which hydrolyzes N-acetylchito-oligosaccharide as substrate. The lysozyme is chitinase, chitobiase, etc. They decompose N-acetyl chito-oligosaccharide to N-acetyl-D-glucosamine.
Japanese Patent No. 94,032,605, May 2, 1994, discloses chitinase-producing Streptomyces microorganism, used for the production of chitin degradation products. A microorganism of Streptomyces produces chitinase. Preferable strains are Streptomyces sp. KE-406 (FERM P-8642), Streptomyces sp. KE-902 (FERM P-8643) and Streptomyces sp. KE-3332 (FERM P-8644). They are all isolated from soil. Chitinase with high activity is produced by culture of a strain of Streptomyces. By using the chitinase, chitinase degradation products, e.g. N-acetyl glucosamine and chitooligosaccharide are produced efficiently.
Chemical Abstract 115:230433 discloses that chitinolytic enzymes containing chitinase and .beta.-N-acetyl-hexosaminidase from Nocardia orientalis IFO 12806 were immobilized on various carriers by different methods for the continuous production of N-acetyl glucosamine. The immobilized enzyme containing both enzymes prepared by physical adsorption to tannin-chitosan was very useful for N-acetyl glucosamine production.